Mutation within the connexin 26 gene responsible for prelingual non-syndromic deafness and method of detection

ABSTRACT

A purified polynucleotide having a chain of nucleotides corresponding to a mutated sequence, which in a wild form encodes a polypeptide implicated in hereditary sensory defect, wherein said mutated purified polynucleotide presents a mutation responsible for prelingual non-syndromic deafness selected from the group consisting of a specific deletion of at least one nucleotide.

This application is a continuation of application Ser. No. 10/278,089,filed Oct. 23, 2002, now U.S. Pat. No. 7,258,975, which is acontinuation of Ser. No. 09/485,415, filed May 3, 2000, now U.S. Pat.No. 6,485,908, which is a 371 of PCT/EP98/05175, filed Aug. 14, 1998,which claims the benefit of priority to U.S. Provisional Application No.60/055,863, filed Aug. 15, 1997, all of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

The present invention concerns a mutation responsible for autosomalprelingual non-syndromic deafness and a method for the detection of thishereditary sensory defect for homozygous and heterozygous individuals.The invention concerns more particularly a specific deletion of at leastone nucleotide in the connexin 26 (Cx 26) gene and especially in aguanosine rich region, notably between the nucleotides 27 and 32. Theinvention is also directed to the use of polynucleotide, or fragmentsthereof, for example as tools useful for the in vitro detection of amutation of a gene belonging to the Cx26 gene family.

Profound or severe prelingual deafness affects one child in a thousandin developed countries (Morton N E. Genetic epidemiology of hearingimpairment. In Genetics of hearing impairment. (The New York Acad Sci,New York 1991; 630:16-31). It is a major handicap as it impedes languageacquisition.

According to studies performed in a U.S. population of children withnon-syndromic (isolated) prelingual deafness and in whom an obviousenvironmental cause has been excluded, it is estimated that up totwo-thirds of the cases have a genetic basis (Marazita M L, Ploughman LM, Rawlings B, Remington E, Amos K S, Nance W E. Genetic epidemiologicalstudies of early-onset deafness in the U.S. school-age population. Am JMed Genet. 1993; 46:486-91). These forms are mainly sensorineural andare almost exclusively monogenic. The major mode of inheritance isautosomal recessive (DFNB), involving 72% to 85% of cases, this fractionincreasing to 90% when only profound deafness is taken into account.Autosomal recessive prelingual deafness is known to be geneticallyhighly heterogeneous. Estimates of the number of DFNB loci vary fromthirty to one hundred (Petit C. Autosomal recessive non-syndromalhearing loss. In Genetics and Hearing Impairment. Martini A, Read A P,Stephens D, eds (Whurr, London) 1996; 197-212), for a review), of whichfourteen have so far been mapped to the human chromosomes (Petit C.Genes responsible for human hereditary deafness: symphony of a thousand.Nature Genet. 1996; 14:385-91) for review, (Verhoeven K, Van Camp G,Govaerts P J, et al. A gene for autosomal dominant non-syndromic hearingloss (DFNA12) maps to chromosome 11q22-24. Am J Hum Genet. 1997;60:1168-74 and Campbell D A, McHale D P, Brown K A, et al. A new locusfor non-syndromal autosomal recessive sensorineural hearing loss(DFNB16) maps to human chromosome 15q21-q22. J Med Genet. 1997; inpress).

A majority of the families attending genetic counseling clinics consistof normal hearing parents with a single deaf child who wish to know therisk of recurrence of the defect. In most cases, given the major role ofenvironmental causes of prelingual deafness, it is not usually possibleeven to recognize whether the hearing loss is of genetic origin. Geneticcounseling in such families would be greatly improved by an ability todetect DFNB mutations. In this respect, the high genetic heterogeneityof the condition represents a major obstacle.

After the initial identification of the DFNB1 locus on 13q11 in a largeconsanguineous Tunisian family (Guilford P, Ben Arab S, Blanchard S, etal. A non-syndromic form of neurosensory, recessive deafness maps to thepericentromeric region of chromosome 13q. Nature Genet. 1994; 6:24-8),two studies performed on New Zealand/Australian families (Maw M A,Allen-Powell D R, Goodey R J, et al. The contribution of the DFNB1 locusto neurosensory deafness in a Caucasian population. Am J Hum Genet.1995; 57:629-35), and on Italian/Spanish families (Gasparini P, EstivillX, Volpini V, et al. Linkage of DFNB1 to non-syndromic neurosensoryautosomal-recessive deafness in Mediterranean families. Eur J Hum Genet.1997; 5:83-8) suggested that this locus might be a major contributor toprelingual deafness in these populations, although individual lod scoresobtained in these families were not significant owing to the small sizeof these families.

Recently, the Cx26 gene, which encodes a gap junction protein, connexin26, has been shown to underlie DFNB1 deafness. Two different G→Asubstitutions resulting in premature stop codons in three DFNB1 linkedconsanguineous Pakistani families have been reported (Kelsell D P,Dunlop J, Stevens H P, et al. Connexin 26 mutations in hereditarynon-syndromic sensorineural deafness. Nature 1997; 387:80-3). These twosubstitutions were identified, respectively, at codon 77 and at codon24. This result has offered the opportunity directly to assess thishypothesis.

The difficulties encountered in genetic counseling for prelingualnon-syndromic deafness due to the inability to distinguish genetic andnon-genetic deafness in the families presenting a single deaf child wasone of the reasons that led the inventors to undertake acharacterization of the spectrum and prevalence of mutations present inthe Cx26 gene in 35 families from several parts of the world withautosomal recessive prelingual deafness.

SUMMARY OF THE INVENTION

The determination of a mutation in the Cx26 gene has notably renderedpossible the use of a detection probe as a tool for the identificationof a specific form of autosomal prelingual non-syndromic deafness, andmore particularly the useful role of a newly identified 30delG (a Gdeletion at position 30; position 1 being the first base of theinitiator codon) mutation in such families. This invention establishesthat the contribution of the DFNB1 locus predominantly resultsessentially from the 30delG mutation. It is now believed that the 30delGaccounts for about three-quarters of all recessive DFNB1 mutations.

The invention is thus intended to provide a purified polynucleotidehaving a chain of nucleotides corresponding to a mutated sequence, whichin a wild form encodes a polypeptide implicated in hereditary sensorydefect. The mutated purified polynucleotide presents a mutationresponsible for prelingual non-syndromic deafness.

The invention also provides oligonucleotides comprising of 15 to 50consecutive nucleotides of the mutated purified polynucleotide that areuseful as primers or as probes.

In addition, the invention aims to supply a method and a kit for thedetection of the hereditary sensory defect for homozygous asheterozygous individuals.

According to the invention, the purified polynucleotide having a chainof nucleotides corresponding to a mutated sequence, which encodes in awild form a polypeptide implicated in hereditary sensory defect,presents a mutation responsible for prelingual non-syndromic deafnessselected from the group consisting of a specific deletion of at leastone nucleotide.

By mutation, according to the invention it means a specific deletion ofat least one nucleotide. Thus, a mutated sequence means a polynucleotidesequence comprising at least a mutation.

A chain of nucleotides, according to the invention, means apolynucleotide, which encodes not necessarily a polypeptide, but whichpresents between 27 and 2311 nucleotides linked together.

The invention particularly concerns a purified polynucleotide wherein,the specific mutation is a deletion located in a region encodingconnexin 26 of chromosome 13q11-12, preferably located in a guanosinerich region starting at nucleotide 27 preferably at nucleotide 30, andextending to nucleotide 32 or nucleotide 35, all the recited nucleotidesbeing inclusive. More particularly according to the invention, thespecific deleted purified polynucleotide encodes for a truncatedpolypeptide.

By truncated polypeptide, according to the invention it means a fragmentof the polypeptide, which does not present the properties of the wildform of the polypeptide either in length, in amino acid composition, orin functional properties.

A preferred embodiment of a specific deletion is a guanosine deletion atposition 30, also called “30delG mutation”. Another preferred embodimentof the specific deletion is a 38 by deletion beginning at position 30.

The invention also includes a purified polynucleotide, which hybridizesspecifically with any one of the polynucleotides as defined above underthe following stringent conditions: at low temperatures between 23° C.and 37° C., in the presence of 4×SSC buffer, 5×Denhardt's solution,0.05% SDS, and 100 μg/ml of salmon sperm DNA. (1×SSC corresponds to 0.15M NaCl and 0.05M sodium citrate; 1×Denhardt's solution corresponds to0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serumalbumin).

The invention also concerns an oligonucleotide useful as a primer or asa probe comprising 15 to 50 consecutive nucleotides of thepolynucleotide according to any one of the polynucleotides as definedabove. The oligonucleotide sequence is selected from the followinggroup:

A first couple:

(SEQ ID No. 1) 5′-TCTTTTCCAGAGCAAACCGCC-3′ (SEQ ID No. 2)5′-TGAGCACGGGTTGCCTCATC-3′.

The length of the PCR product has been obtained from 285 by in length;

A second couple allowing to explore the other part of the reading frame:

(SEQ ID No. 3) 5′-GACACGAAGATCAGCTGCAG-3′ (SEQ ID No. 4)5′-CCAGGCTGCAAGAACGTGTG-3′

A third couple:

(SEQ ID No. 9) 5′-CTAGTGATTCCTGTGTTGTGTGC-3′; and (SEQ ID No. 10) 5′ATAATGCGAAAAATGAAGAGGA-3′ and

A fourth couple:

(SEQ ID No. 14) 5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCCTAGTGATTCCTGTGTTGTGTGC-3′; and (SEQ ID No. 10) 5′ ATAATGCGAAAAATGAAGAGGA-3′.

Another oligonucleotide useful as a probe is selected from the followinggroup:

(SEQ ID No. 5) 5′-AGACGATCCTGGGGGTGTGAACAAA-3′ (SEQ ID No. 6)5′-ATCCTGGGGGTGTGA-3′ (SEQ ID No. 7) 5′-AGACGATCCTGGGGGCTCACCGTCCTC-3′.

In addition, the invention concerns a method for the detection of anhereditary sensory defect, namely autosomal prelingual non-syndromicdeafness, for homozygous as heterozygous individuals in a biologicalsample containing DNA, comprising the steps of

a) bringing the biological sample into contact with a oligonucleotideprimers as defined above, the DNA contained in the sample having beenoptionally made available to hybridization and under conditionspermitting a hybridization of the primers with the DNA contained in thebiological sample;

b) amplifying the DNA;

c) revealing the amplification products;

d) detecting the mutation.

Step d) of the above-described method may comprise a Single-StrandConformation Polymorphism (SSCP), a Denaturing Gradient GelElectrophoresis (DGGE) sequencing (Smith, L. M., Sanders, J. Z., Kaiser,R. J., Fluorescence detection in automated DNA sequence analysis. Nature1986; 321:674-9); a molecular hybridization capture probe or atemperature gradient gel electrophoresis (TGGE).

Step c) of the above-described method may comprise the detection of theamplified products with an oligonucleotide probe as defined above.

According to the invention, a biological sample can be a blood sampleextracted from people suffering from any kind of deafness with anycriteria as follows: neurosensorial or mixed isolated deafness, advancedor not, at any degree of severity, concerning familial or sporadic case,or individuals exposed to noise, or individuals suffering from a lowacoustic, or individuals susceptible to carry an anomaly in the gene, orfrom an embryo for antenatal diagnostic.

Another aim of the invention comprises a method for the detection of anhereditary sensory defect, the autosomal prelingual non-syndromicdeafness, for homozygous and heterozygous individuals in a biologicalsample containing DNA, comprising the steps of:

a) bringing the biological sample into contact with an oligonucleotideprobe according to the invention, the DNA contained in the sample havingbeen optionally made available to hybridization and under conditionspermitting a hybridization of the primers with the DNA contained in thebiological sample; and

b) detecting the hybrid formed between the oligonucleotide probe and theDNA contained in the biological sample.

Step b) of the above-described method may consist in a single-strandconformation. Polymorphism (SSCP), a denaturing gradient gelelectrophoresis (DGGE) or amplification and sequencing.

The invention also includes a kit for the detection of an hereditarysensory defect, the autosomal prelingual non-syndromic deafness, forhomozygous as heterozygous individuals, said kit comprising:

a) oligonucleotides according to the invention;

b) the reagents necessary for carrying out DNA amplification; and

c) a component that makes it possible to determine the length of theamplified fragments or to detect a mutation.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be more described in greater detail by reference tothe drawings in which:

FIG. 1 depicts the results of temperature gradient gel electrophoresisfor detection of mutants in which:

Lanes 1 and 2: DNA from normal patients.

Lanes 3 and 4: DNA from homozygous patients with 30delG mutation.

Lanes 5 and 6: DNA from heterozygous patients.

Lane 7: PCR control without DNA.

Lane 8: PCR fragment amplified from a normal DNA and hybridized with astandard DNA fragment harboring the 30delG mutation.

Lane 9: PCR fragment amplified from a mutant homozygous DNA andhybridized with a normal standard DNA fragment harboring the guanine 30.

DETAILED DESCRIPTION OF THE INVENTION

Prelingual non-syndromic (isolated) deafness is the most frequenthereditary sensory defect in children. The inheritance in most isautosomal recessive. Several dozens of genes might be involved, only twoof which, DFNB1 and DFNB2, have so far been identified (Kelsell, D. P.,et al., Connexin 26 mutations in hereditary non-syndromic sensorineuraldeafness. Nature 1997; 387:80-3; Liu, X-Z, et al., Mutations in themyosin VIIA gene cause non-syndromic recessive deafness, Nature Genet.1997; 16:188-90; and Weil, D., et al., The autosomal recessive isolateddeafness, DFNB2, and the Usher 1B syndrome are allelic defects of themyosin-VIM. Nature Genet. 1997; 16:191-3). A search was made searchedfor mutations in the gene encoding connexin 26, Cx26, which has recentlybeen shown to be responsible for DFNB1. Mutation analysis of Cx26 wasperformed by PCR amplification on genomic DNA and sequencing of thesingle coding exon.

Example 1 Patients

Thirty-five affected families from various geographical regions, mainlyFrance, New Zealand and Australia, Tunisia and Lebanon, were studied.They could be classified into three categories: (1) consanguineousfamilies each having a significant linkage to the DFNB1 locus; (2) smallnon-consanguineous families in which linkage analysis was compatiblewith the involvement of DFNB1; and (3) small families in which nolinkage analysis had been undertaken.

The first category consists of six large families living ingeographically isolated regions. Five were from Tunisia, two from thenorth and three from the south. Linkage to the DFNB1 locus in the twofamilies from northern Tunisia (families 20 and 60) had previously beenreported (Guilford P, Ben Arab S, Blanchard S, et al., A non-syndromicform of neurosensory, recessive deafness maps to the pericentromericregion of chromosome 13q. Nature Genet. 1994; 6:24-8); the threefamilies from southern Tunisia (S15, S19 and ST) and the family fromLebanon (LH) comprise total of three, five, two, and five deaf children,respectively, the deafness being of severe or profound degree. Themarriages were between first cousins (S15, ST and LH) and between firstand second cousins (S19). Linkage analysis of these six familiesresulted in individual lod scores ranging from 2.5 to 10 withpolymorphic markers from the DFNB1 region (D13S175, D13S141, D13S143 andD13S115).

The second category of patients comprises seven New Zealand familieswith at least two deaf siblings (families 51, 1160, 1548, 1608, 1773,1873, 1877) and one Australian (9670) family. Family 1608 was atypicalin that four siblings sharing the same DFNB1 marker haplotypes had amild to moderate deafness (severe at high frequency), with the child ofone of them being profoundly deaf. In family 1873, the unrelated parents(individuals 11.2 and 11.3) were deaf as well as their two children, andwe have therefore considered this as two families, bringing to nine thetotal of independent families. Apart from families 1608 and 1873, noparent acknowledged any hearing impairment. These nine families showedcosegregation between deafness and polymorphic markers of the DFNB1region with maximum individual lod scores ranging from 0.6 to 1.2. Tenother families in the original study of Maw et al. (Maw M A,Allen-Powell D R, Goodey R J, et al. The contribution of the DFNB1 locusto neurosensory deafness in a Caucasian population. Am J Hum Genet.1995; 57:629-35) had shown no cosegregation and one other cosegregatingfamily was not tested for Cx26 mutations. The New Zealand families wereall of Caucasian origin with no known Polynesian admixture. According tothe antecedent family names, the ancestral proportion among the familiesreflected that of the general Caucasian New Zealand population with thegreat predominance being of Anglo-Celtic patrimony and a small fractiondue to migration from continental Europe. Neither parentalconsanguinity, nor links between any of the families were recognized. Inthe Australian case, the father was from Northern Ireland and the motherfrom Yorkshire, England.

The third category is composed of nineteen families living in France andtwo in New Zealand, each with at least two children having a severe toprofound deafness. No parent acknowledged any hearing impairment, exceptfor the mother in family P16 and the father in family P17 who hadmoderate and progressive high-frequency hearing loss. Five of thesefamilies had foreign ancestors from Lebanon (family P3), Turkey (familyP4), Portugal (family P9), Algeria (family P14) and Poland (father infamily P16). In two of the families (P7 and P14), the parents weredistantly related.

Example 2

Amplification of the coding exon of Cx26 PCRs were carried out ongenomic DNA using a set of primers that allowed the amplification of theentire coding sequence of the Cx26 gene, which consists of a singlecoding exon (Kelsell D P, Dunlop J, Stevens H P, et al. Connexin 26mutations in hereditary non-syndromic sensorineural deafness. Nature1997; 387: 80-3). Primer sequences were as follows:

(SEQ ID No. 1) 5′-TCTTTTCCAGAGCAAACCGCC-3′ and (SEQ ID No. 2)5′-TGAGCACGGGTTGCCTCATC-3′.

PCR conditions were: 35 cycles of 95° C., 1 min; 58° C., 1 min; 72° C.,2 min. The PCR product obtained was 777 by in length.

Example 3 DNA Sequencing

Sequencing of the PCR products was performed as previously described(Smith L M, Sanders J Z, Kaiser R J, et al., Fluorescence detection inautomated DNA sequence analysis, Nature 1986; 321:674-9) using thedideoxy chain terminator method on an Applied Biosystems DNA sequencerABI373 with fluorescent dideoxynucleotides. The primers used were thesame as those for the PCR amplification plus two internal primers

(SEQ ID No. 3) 5′-GACACGAAGATCAGCTGCAG-3′ and (SEQ ID No. 4)5′-CCAGGCTGCAAGAACGTGTG-3′.

Example 4 Mutations in Consanguineous Tunisian and Lebanese DFNB1Families

In these families the involvement of the DFNB1 locus could bedemonstrated by linkage analysis. In four of the five families fromTunisia (S15, S19, 20, and 60) and in the Lebanese family (LH), the samemutation was detected in all affected children on both Cx26 alleles,namely, a deletion of a guanosine (G) in a sequence of six G extendingfrom position 30 to 35 (position 1 being the first base of the initiatorcodon) (Table 1). This mutation is hereafter referred to as 30delGmutation according to the nomenclature proposed by Beaudet and Tsui((Beaudet A L, Tsui L-C. A suggested nomenclature for designatingmutations, Hum Mutation 1993; 2: 245-8)). It creates a frameshift, whichresults in a premature stop codon at nucleotide position 38. Themutation segregating in the fifth family from Tunisia (ST) wasidentified as a G to T transversion at nucleotide position G39 creatinga premature stop codon (GAG→TAG) at codon 47, and was designated E47X.In each family, normal hearing parents were found to be heterozygous forthe corresponding mutation.

Example 5 Mutations in Small Nonconsanguineous New Zealand andAustralian Families Consistent with DFNB1 Linkage

In these families, segregation analysis has previously been reported ascompatible with the involvement of the DFNB1 locus (Maw M A,Allen-Powell D R, Goodey R J, et al. The contribution of the DFNB1 locusto neurosensory deafness in a Caucasian population. Am J Hum Genet.1995; 57: 629-35). The deaf individuals from five of the nine families(51, 1160, 1608 (III.20), 1873 (II.3) and 1877) were homozygous for the30delG mutation. The deaf children from family 1773 were heterozygousfor 30delG. Deaf individual II.2 from family 1873 (see “subjects” andTable 1) was heterozygous for a deletion of 38 by beginning atnucleotide position G30, designated 30del38. No other mutation wasdetected in the deaf children of family 1773 and the deaf individual(II.2) in family 1873. Nevertheless, in this last individual, a deletionof the polymorphic marker immediately proximal to the Cx26 gene (locusD13S175) had previously been observed (Maw M A, Allen-Powell D R, GoodeyR J, et al. The contribution of the DFNB1 locus to neurosensory deafnessin a Caucasian population. Am J Hum Genet. 1995; 57: 629-35), which mayindicate that a DNA rearrangement has impaired the functioning of theother Cx26 allele of the gene in cis. In family 9670, compoundheterozygosity for a missense mutation (R184P) and an in framesingle-codon deletion (delE138) was observed in affected siblings. Inonly one family (1548) was no Cx26 mutation detected. Results aresummarized in Table 1.

Example 6 Mutations in Small Families Uncharacterized for DFNB1 LinkageLiving in France and New Zealand

Nineteen families (P1 to 17, L14190 and L13131) living in France and twoin New Zealand (families 1885 and 2254) were studied. In these families,cosegregation of the deafness with polymorphic markers had not beenanalysed. Deaf children from six of the twenty-one families (P1, P3, P5,P9, P10, and P16) were found to be homozygous for the mutation 30delG.In five additional families (P6, P11, P14, P17, and 1885), deaf childrenwere heterozygous for this mutation; no other mutation was detected inthese families. In the ten remaining families, no mutation in the Cx26gene was found.

Example 7 Molecular Hybridization Using Allele-Specific Capture Probes

Molecular hybridization capture probe (see, e.g., D. Chevrier et al. PCRproduct quantification by non-radioactive hybridization procedures usingan oligonucleotide covalently bound to microwells. Molecular andCellular Probes 1993; 7: 187-197 and D Chevrier et al. Rapid detectionof Salmonella subspecies I by PCR combined with non-radioactivehybridization using covalently immobilized oligonucleotide on amicroplate. FEMS Immunology and Medical Microbiology 1995; 10: 245-252each of which is incorporated by reference herein) permit specificdetection of the 30delG mutation. The technique has been adapted topermit rapid diagnosis of prelingual non-syndromic deafness caused bythe 30delG mutation. The technique provides certain advantages in aclinical setting because it uses stable, nonradioactive molecules, itcan be easily automated, and it is well adapted to large scale analysis.

Using primers designed for PCR amplification, the region of interest inthe Cx26 gene is amplified from genomic DNA samples. The primersequences are as follows:

(SEQ ID No. 9) CONN3: 5′-CTAGTGATTCCTGTGTTGTGTGC-3′ (SEQ ID No. 10)CONN4: 5′ ATAATGCGAAAAATGAAGAGGA-3′

PCR is performed with the CONN3 (SEQ ID No. 9) and CONN4 (SEQ ID No. 10)primers (1 μM each), an aliquot of the DNA to be analyzed (2 μl, 100-300ng), 1.5 mM MgCl₂, 200 μM dNTP, and Taq polymerase. The amplificationprogram consists of the following steps: 1) 95° C., 5 min; 2) additionof enzyme, 95° C., 1 min; 3) 60° C., 1 min (ramp rate=0.25° C./s); 4)72° C., 1 min; 5) repeat steps 2 to 4 for 40 cycles; and 6) 72° C., 10min. PCR products are verified by a rapid gel electrophoresis.

The amplified PCR product contains either the normal or the mutant Cx26sequence. To distinguish between the normal and mutant sequence, twocapture probes are designed. The sequences of these two capture probesare as follows:

For detection of normal sequence:

(SEQ ID No. 11) CONN6: 5′-AAAAAAAATCCTGGGGGGTGTG-3′

For detection of mutant sequence:

(SEQ ID No. 12) CONN7: 5′-AAAAAAAATCCTGGGGGTGTGA-3′Each capture probe must be 22 nucleotides long. Furthermore, to beefficient, the capture probe must include an A₇ spacer at its 5′ end anda hybridization region of 15 bases. Such a capture probe is able tospecifically differentiate the mutant sequence from the normal sequence.Thus, CONN6 (SEQ ID No. 11) is designed to specifically hybridize withthe normal sequence, whereas CONN7 (SEQ ID No. 12) is designed tospecifically hybridize with the mutant sequence.

Before attaching the capture probes to a microtiter plate, they arephosphorylated at their 5′ ends. The phosphorylation is carried out for1 hour at 37° C. in presence of 20 nmoles of CONN6 (SEQ ID No. 11) orCONN7 (SEQ ID No. 12) oligonucleotides, 100 μM ATP, 10 units T4polynucleotide kinase in 200 μl of buffer (50 mM Tris-HCl pH 7.4; 10 mMMgCl₂ 5 mM dithiothreitol; and 1 mM spermidine). The mixture is heatedfor 10 min. at 68° C. to inactivate the T4 polynucleotide kinase, thenthe oligonucleotide is precipitated by adding 145 μl of 10 M CH₃COONH₄,15 μl H₂O, and 800 μl iced ethanol. After a 30 min. incubation in ice,the mixture is centrifuged for 20 min. at 12,000×g at 4° C. Theresulting pellet is washed with 500 μl iced ethanol (70%) and dissolvedin 800 μl of TE buffer. The phosphorylated oligonucleotide concentrationis determined by optical density at 260 nm.

Before attaching the phosphorylated oligonucleotides to microplates,they are denatured by heating at 95° C. for 10 min. and rapidly cooledin ice to avoid the formation of secondary structure. 500 ng ofphosphorylated CONN6 (SEQ ID No. 11) or CONN7 (SEQ ID No. 12) and 1 μlof 1 M 1-methylimidazole, pH 7, is added to each well of a microplate,which is kept on ice. The total volume of each well is adjusted to 70 μlwith distilled water, before adding 30 μl of a cold,1-ethyl-3(3-dimethylaminopropyl) carbodiimide solution (167 mM). Themicroplate is covered and incubated for 5 hours at 50° C. in anincubator (Thermomix® from Labsystems). After the 5-hour incubation, themicroplate is washed three times with a warm solution (50° C.) of 0.4 NNaOH containing 0.25% SDS. The microplate is incubated for 5 min. withthe same warm solution and washed again with warm NaOH/SDS (50° C.).Finally, the microplate is washed five times with TE buffer. The coatedmicroplate can be kept several months at 4° C., if the wells are filledwith TE buffer.

The amplified sequences from the genomic DNA samples are incubated witha biotinylated detection probe in the coated microplates. Unlike thecapture probes, which are allele specific, the detection probe canhybridize with both the normal and mutant sequences. The sequence of thedetection probe is:

(SEQ ID No. 13) CONN12: 5′-CAGCATTGGAAAGATCTGGCTCA-3′.The amplified sequences and the detection probe, which is biotinylatedat its 5′ end, are denatured directly in the microplates by successivelyadding to each well: 95 μl of water, 5 μl of PCR reaction, 40 μl ofbiotinylated probe (SEQ ID No. 13) at 22 nM diluted in water, and 14 μl1 N NaOH. After 10 min., 21 μl of 1 M NaH₂PO₄ and 1% Sarkosyl is addedto each well to bring the total volume to 175 μl per well. The finalconcentration of the detection probe is 5 nM. The microplate is coveredand incubated overnight at 40° C. in an incubator (Thermomix® fromLabsystems) and then extensively washed (5 times) with TBS-Tween toremove the excess biotinylated probe (SEQ ID No. 13).

An immunoenzymatic method is used to detect the hybridized probe. Eachwell receives 100 μl of the conjugate (Extravidine—alkaline phosphatase,Sigma E-2636) diluted 1/4000 in TBS-BSA-Tween. The microplate is coveredand incubated for 1 hour at 25° C. Following the incubation, themicroplate is washed 5 times with TBS-Tween. Then 200 μl of preheated(37° C.) substrate (7.5 mg para-nitro-phenyl-phosphate in 20 ml of thefollowing buffer: 1 M diethanolamine pH 9.8 containing 1 mM MgCl₂) areadded to each well. The microplate is covered and incubated for 3 hoursat 37° C. The absorbance is measured at 405 nm to determine the specificsignal and at 630 nm to determine the background noise.

The hybridization ratio (R) between the signal obtained with CONN6 (SEQID No. 11) probe (normal sequence) and that obtained with CONN7 (SEQ IDNo. 12) probe mutant sequence) is calculated. The calculated R valuesare used to determine the genotypes of the sample DNA as follows:homozygous for the normal Cx26 sequence (R 2), heterozygous for the30delG mutation (0.5<R<2), and homozygous for the 30delG mutation (R S0.5). The range of the hybridization ratio (R) can be slightly modifiedwhen the number of samples increases. The following table represents anexample of results obtained with 39 samples.

Hybridization ratio (R) Genotype: Normal Homozygous 30delG Heterozygous5.96 0.48 1.33 5.43 0.17 1.13 3.39 0.21 0.73 4.14 0.16 0.63 4.09 0.281.4 2.76 0.13 0.73 2.2 0.21 0.76 3.97 0.4 0.73 4.07 1.06 3 2.76 3.663.87 3.92 3.26 5.17 2.74 4.51 6.3 3.49 4.05 3.17 Number 22 8 9 Meanvalue 3.91 0.26 0.94 Standard 1.06 0.12 0.29 deviation Range (6.3-2.2)(0.48-0.13) (1.4-0.63)

Example 8 Temperature Gradient Gel Electrophoresis

Temperature gradient gel electrophoresis (TGGE) permits the detection ofany type of mutation, including deletions, insertions, andsubstitutions, which is within a desired region of a gene. (See, e.g. D.Reiner et al. Temperature-gradient gel electrophoresis of nucleic acids:Analysis of conformational transitions, sequence variations andprotein-nucleic acid interactions. Electrophoresis 1989; 10: 377-389; E.P. Lessa and G. Applebaum Screening techniques for detecting allelicvariation in DNA sequences. Molecular Ecology 1993; 2: 119-129 and A. L.Börresen-Dale et al. Temporal Temperature Gradient Gel Electrophoresison the D code™ System. Bio-Rad US/EG Bulletin 2133; the entiredisclosure of each publication is incorporated by reference herein.)However, TGGE does not permit one to determine precisely the type ofmutation and its location.

As in the previously described molecular hybridization technique, theregion of interest in the Cx26 gene is first amplified from genomic DNAsamples by PCR. The primer sequences are as follows:

(SEQ ID No. 14) CONN2: 5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCCTAGTGATTCCTGTGTTGTGTGC-3′ (SEQ ID No. 10) CONN4: 5′ATAATGCGAAAAATGAAGAGGA-3′PCR is performed with 1 μM of the CONN2 (SEQ ID No. 14) primer, whichhas a GC clamp at its 5′ end, and 1 μM of the CONN4 (SEQ ID No. 10)primer, an aliquot of the DNA to be analyzed (2 μl, 100-300 ng), 1.5 mMMgCl₂, 200 μM dNTP, and Taq polymerase. The amplification programconsists of the following steps: 1) 95° C., 5 min; 2) addition ofenzyme, 95° C., 1 min; 3) 60° C., 1 min (ramp rate=0.25° C./s); 4) 72°C., 1 min; 5) repeat steps 2 to 4 for 40 cycles; and 6) 72° C., 10 min.

Analyzing these PCR amplification fragments by TGGE can differentiatebetween homozygous (normal or mutant) samples, which produce a singleband on a gel, and heterozygous samples, which produce three bands.However, differentiating between genomic samples that are homozygous forthe normal sequence and genomic samples that are homozygous for the30delG mutants requires an additional step.

To differentiate normal homozygous versus mutant homozygous samples, analiquot of the amplified PCR product is mixed with either a known,normal homozygous sample or a known, 30delG mutant homozygous sample andanalyzed for heteroduplex formation. If the amplified PCR productderives from a normal, homozygous sample, it will form a heteroduplexwith the known, 30delG mutant homozygous sample. On the other hand, ifthe amplified PCR product derives from a mutant, homozygous sample, itwill form a heteroduplex with the known, normal homozygous sample. Topromote heteroduplex formation in these mixtures, they are denatured at95° C. for 5 min, followed by a renaturation step at 60° C. for 45 min.

The PCR fragments from the initial amplification and those that aresubjected to the additional heating steps to permit heteroduplexformation are analyzed on a 10% polyacrylamide gel containing 7 M urea.By way of example, a 30 ml gel is prepared by combining the followingingredients:

-   -   12.6 g urea    -   0.75 ml 50×TAE    -   7.5 ml acrylamide:bisacrylamide (37.5:1) at 40%    -   water to bring volume to 30 ml    -   30 μl Temed (added extemporaneously)    -   300 μl 10% ammonium persulfate (added extemporaneously).        After adding the Temed and ammonium persulfate, the gel is        poured between two glass plates (Dcode Universal Mutation        Detection System® from BIORAD) and allowed to polymerize for 1        hour.

An aliquot (7.5) of the PCR mixture is mixed with 7.5 μl of 2× samplesolution (2 mM EDTA pH 8; 70% glycerol; 0.05% xylene cyanol; 0.05%bromophenol blue), and introduced into a gel well. Electrophoresis isperformed for 4-5 hours at 150V in 1.25×TAE buffer with a temperaturegradient ranging from 61° C. to 62° C. at a rate of 0.2° C. per hour.Following electrophoresis, the gel is incubated for 6 min. in 1.25×TAEcontaining 25 μg/ml ethidium bromide. Excess ethidium bromide is removedby a 20 min. wash in 1.25×TAE, and the DNA fragments are visualized witha UV transilluminator.

A typical TGGE result is represented in FIG. 1. The amplified DNA fromhomozygous patients (normal or mutant) produces only one band. Theamplified DNA from heterozygous patients results in three differentfragments in the polyacrylamide gel. The more intense band, whichmigrates more rapidly, corresponds to both homoduplexes, which cannot beseparated in this gel. The other two bands, which migrate more slowly,correspond to both kinds of heteroduplexes.

The DNA of normal homozygous patients can be differentiated from the DNAof mutant homozygous patients by analyzing the PCR fragments that weresubjected to the conditions that permitted heteroduplex formation.Heteroduplexes form when the PCR amplified fragment from a normalhomozygous genome is mixed with sequences from a known, mutanthomozygous genome, or when the PCR amplified fragment from a mutanthomozygous genome is mixed with sequences from a known, normalhomozygous genome. These heteroduplexes are visible by TGGE analysis.Consequently, the DNA of normal and mutant homozygous patients can beeasily differentiated by this technique using the primers described inthe present study.

In all the known DFNB1 families (6/6), in all but one (8/9) of theputatively DFNB1-linked families, and in about half (11/21) of thefamilies not tested for DFNB1 linkage, a mutation in Cx26 was detected.Furthermore, of the 44 chromosomes reckoned to be independent upon whicha Cx26 mutant allele was identified or inferred, 33 (75%) were found tocarry the same deletion of a guanosine, G, at position 30 (30delG).

Cx26 mutations represent a major cause of recessively inheritedprelingual deafness and would be implicated in about half of cases inthe examined populations. In addition, one specific mutation, 30delG,accounts for the majority (about three-quarters in our series) of theCx26 mutant alleles.

The wild type connexin 26 gene published in LEE S. W. et al. (1992) J.Cell Biol. 118: 1213-1221 has the following sequence:

   1 GATTTAATCC TATGACAAAC TAAGTTGGTT CTGTCTTCAC CTGTTTTGGT   51GAGGTTGTGT AAGAGTTGGT GTTTGCTCAG GAAGAGATTT AAGCATGCTT  101GCTTACCCAG ACTCAGAGAA GTCTCCCTGT TCTGTCCTAG CTATGTTCCT  151GTGTTGTGTG CATTCGTCTT TTCCAGAGCA AACCGCCCAG AGTAGAAGAT  201GGATTGGGGC ACGCTGCAGA CGATCCTGGG GGGTGTGAAC AAACACTCCA  251CCAGCATTGG AAAGATCTGG CTCACCGTCC TCTTCATTTT TCGCATTATG  301ATCCTCGTTG TGGCTGCAAA GGAGGTGTGG GGAGATGAGC AGGCCGACTT  351TGTCTGCAAC ACCCTGCAGC CAGGCTGCAA GAACGTGTGC TACGATCACT  401ACTTCCCCAT CTCCCACATC CGGCTATGGG CCCTGCAGCT GATCTTCGTG  451TCCAGCCCAG CGCTCCTAGT GGCCATGCAC GTGGCCTACC GGAGACATGA  501GAAGAAGAGG AAGTTCATCA AGGGGGAGAT AAAGAGTGAA TTTAAGGACA  551TCGAGGAGAT CAAAACCCAC AAGGTCCGCA TCGAAGGCTC CCTGTGGTGG  601ACCTACACAA GCAGCATCTT CTTCCGGGTC ATCTTCGAAG CCGCCTTCAT  651GTACGTCTTC TATGTCATGT ACGACGGCTT CTCCATGCAG CGGCTGGTGA  701AGTGCAACGC CTGGCCTTGT CCCAACACTG TGGACTGCTT TGTGTCCCGG  751CCCACGGAGA AGACTGTCTT TCACAGTGTT CATGATTGCA GTGTCTGGAA  801TTTGCATCCT GCTGAATGTC ACTGAATTGT GTTATTTGCT AATTAGATAT  851TGTTCTGGGA AGTCAAAAAA GCCAGTTTAA CGCATTGCCC AGTTGTTAGA  901TTAAGAAATA GACAGCATGA GAGGGATGAG GCAACCCGTG CTCAGCTGTC  951AAGGCTCAGT CGCCAGCATT TCCCAACACA AAGATTCTGA CCTTAAATGC 1001AACCATTTGA AACCCCTGTA GGCCTCAGGT GAAACTCCAG ATGCCACAAT 1051GAGCTCTGCT CCCCTAAAGC CTCAAAACAA AGGCCTAATT CTATGCCTGT 1101CTTAATTTTC TTTCACTTAA GTTAGTTCCA CTGAGACCCC AGGCTGTTAG 1151GGGTTATTGG TGTAAGGTAC TTTCATATTT TAAACAGAGG ATATCGGCAT 1201TTGTTTCTTT CTCTGAGGAC AAGAGAAAAA AGCCAGGTTC CACAGAGGAC 1251ACAGAGAAGG TTTGGGTGTC CTCCTGGGGT TCTTTTTGCC AACTTTCCCC 1301ACGTTAAAGG TGAACATTGG TTCTTTCATT TGCTTTGGAA GTTTTAATCT 1351CTAACAGTGG ACAAAGTTAC CAGTGCCTTA AACTCTGTTA CACTTTTTGG 1401AAGTGAAAAC TTTGTAGTAT GATAGGTTAT TTTGATGTAA AGATGTTCTG 1451GATACCATTA TATGTTCCCC CTGTTTCAGA GGCTCAGATT GTAATATGTA 1501AATGGTATGT CATTCGCTAC TATGATTTAA TTTGAAATAT GGTCTTTTGG 1551TTATGAATAC TTTGCAGCAC AGCTGAGAGA GGCTGTCTGT TGTATTCATT 1601GTGGTCATAG CACCTAACAA CATTGTAGCC TCAATCGAGT GAGACAGACT 1651AGAAGTTCCT AGTTGGCTTA TGATAGCAAA TGGCCTCATG TCAAATATTA 1701GATGTAATTT TGTGTAAGAA ATACAGACTG GATGTACCAC CAACTACTAC 1751CTGTAATGAC AGGCCTGTCC AACACATCTC CCTTTTCCAT GCTGTGGTAG 1801CCAGCATCGG AAAGAACGCT GATTTAAAGA GGTGAGCTTG GGAATTTTAT 1851TGACACAGTA CCATTTAATG GGGAGACAAA AATGGGGGCC AGGGGAGGGA 1901GAAGTTTCTG TCGTTAAAAA CGAGTTTGGA AAGACTGGAC TCTAAATTCT 1951GTTGATTAAA GATGAGCTTT GTCTACCTTC AAAAGTTTGT TTGGCTTACC 2001CCCTTCAGCC TCCAATTTTT TAAGTGAAAA TATAACTAAT AACATGTGAA 2051AAGAATAGAA GCTAAGGTTT AGATAAATAT TGAGCAGATC TATAGGAAGA 2101TTGAACCTGA ATATTGCCAT TATGCTTGAC ATGGTTTCCA AAAAATGGTA 2151CTCCACATAG TTCAGTGAGG GTAAGTATTT TCCTGTTGTC AAGAATAGCA 2201TTGTAAAAGC ATTTTGTAAT AATAAAGAAT AGCTTTAATG ATATGCTTGT 2251AACTAAAATA ATTTTGTAAT GTATCAAATA CATTTAAAAC ATTAAAATAT 2301AATCTCTATA AT

The wild type connexin 26 gene is published in D. T. Kiang et al. (1997)Gene 199 (1-2): 165-171.

TABLE 1 Mutations in the Cx26 coding exon in individuals affected withfamilial forms of prelingual deafness Family (geographical 30delG Otherorigin) mutation mutation Deafness DFNB1-linked families S15 (sTu)homozygous — profound S19 (sTu) homozygous — profound ST (sTu) —homozygous profound E47X 20 (nTu) homozygous — profound 60 (nTu)homozygous — profound LH (Leb) homozygous severe-profound Familiesconsistent with DFNB1 linkage 51 (NZ) homozygous — severe-profound 1160(NZ) homozygous — moderate-severe* 1548 (NZ) — — profound 1608 (NZ)homozygous — profound** 1773 (NZ) heterozygous — profound 1873individual II.3 homozygous — moderate (NZ) 1873 individual II.2 —heterozygous profound (NZ) 30del38 1877 (NZ) homozygous — profound 9670(Aust) delE118/R148P moderate-severe Families uncharac- terized forDFNB1 linkage P1 (Fr) homozygous — severe-profound P2 (Fr) — — profoundP3 (Leb) homozygous — severe-profound P4 (Tur) — — severe-profound P5(Fr) homozygous — profound P6 (Fr) heterozygous — severe-profound P7(Fr) — — moderate P8 (Fr) — — moderate L13131 (Fr) — — profound L14190(Fr) — — mild-moderate P9 (Por) homozygous — severe-profound P10 (Fr)homozygous — severe-profound P11 (Fr) heterozygous — moderate-severe P12(Fr) — — severe-profound P13 (Fr) — — profound P14 (Alg) heterozygous —moderate-severe P15 (Fr) — — severe-profound P16 (mother/Fr, homozygous— severe** father/Pol) P17 (Fr) heterozygous — severe*** 1885 (NZ)heterozygous — profound 2254 (NZ) — — moderate-severe The analysisreported here concerns deaf children of the various families except forfamily 1873 (see patients and methods). *moderate in one ear, severe inthe other ear. **moderate hearing loss in mother (severe at highfrequencies), ***mild hearing loss in father, who are heterozygouscarriers for the 30delG mutation. Geographical origins: (Alg) Algeria,(Aust) Australia, (Fr) France, (Leb) Lebanon, (NZ) New Zealand, (Pol)Poland, (Por) Portugal, (nTu) North Tunisia, (sTu) South Tunisia, (Tur)Turkey

1-28. (canceled)
 29. A method for diagnosing autosomal prelingualnonsyndromic deafness in a human, the method comprising determining thepresence of a mutation identified as a G to T transversion at nucleotideposition G139 creating a premature stop codon (GAG→JAG) at codon 47(designated E47X) of a connexin 26 gene in a DNA sample obtained fromthe human, wherein position 1 is the first base of the initiator codon,and diagnosing autosomal prelingual nonsyndromic deafness.
 30. A methodfor diagnosing deafness in a human, wherein the deafness is associatedwith the presence of a mutation identified as a G to T transversion atnucleotide position G139 creating a premature stop codon (GAG→JAG) atcodon 47 (designated E47X) of a connexin 26 gene, wherein position 1 isthe first base of the initiator codon, the method comprising determiningthe presence of the E47X mutation in a connexin 26 gene present in a DNAsample obtained from the human.
 31. A method for detecting the presenceof a mutation identified as a G to T transversion at nucleotide positionG139 creating a premature stop codon (GAG→JAG) at codon 47 (designatedE47X) of a connexin 26 gene in a biological sample containing DNA,wherein position 1 is the first base of the initiator codon, the methodcomprising: a) bringing the biological sample into contact with a pairof oligonucleotide primers consisting of 15 to 50 nucleotides, whereinthe oligonucleotide primers hybridize under stringent conditions with aregion of interest of the connexin 26 gene comprising a E47X mutation,under conditions permitting hybridization of the oligonucleotide primerswith the DNA contained in the biological sample; b) amplifying theregion of interest of the connexin 26 gene; c) detecting theamplification products; and d) detecting the mutation in theamplification products.
 32. A method for detecting the presence orabsence of a mutation identified as a G to T transversion at nucleotideposition G139 creating a premature stop codon (GAG→JAG) at codon 47(designated E47X) of a connexin 26 gene, wherein position 1 is the firstbase of the initiator codon, the method comprising: a) bringing abiological sample containing DNA from a human into contact with anoligonucleotide consisting of 15 to 50 consecutive nucleotides, whereinthe oligo-nucleotide hybridizes with a connexin 26 gene comprising aE47X mutation, and does not hybridize with a wild-type connexin 26 gene,under conditions permitting hybridization of the oligonucleotide withthe DNA contained in the biological sample; and b) detecting thepresence or absence of hybrids formed between the oligonucleotide andthe DNA contained in the biological sample, thereby indicating thepresence or absence, respectively, of the mutation.
 33. The method ofclaim 31, wherein step d) comprises: a) incubating the amplificationproducts with a labeled detection probe that hybridizes with a region ofinterest of both a normal connexin 26 sequence and a mutant connexin 26sequence comprising a first capture probe that hybridizes with a regionof interest of the normal connexin 26 sequence but does not hybridizewith a region of interest of the mutant connexin 26 sequence; b)incubating the amplification products with the labeled detection probeand a second capture probe that hybridizes with a region of interest ofthe mutant connexin 26 sequence but does not hybridize with a region ofinterest of the normal connexin 26 sequence; c) hybridizing theamplification products with the labeled detection probe and with thefirst capture probe or the second capture probe; and d) comparing thehybridization signal obtained from the first capture probe with thehybridization signal obtained from the second capture probe.
 34. Themethod of claim 33, wherein the detection probe is labeled with biotin.35. The method of claim 33, wherein the first capture probe and thesecond capture probe are bound to a microplate.
 36. The method of claim31, wherein the biological sample is blood.
 37. The method of claim 31,wherein in step d) the mutation is detected by a technique selectedfrom: single-strand conformation polymorphism (SSCP); denaturinggradient gel electrophoresis (DGG); sequencing; and temperature gradientgel electrophoresis (TGGE).
 38. The method of claim 33, wherein thedetection probe is non-radioactively labeled.
 39. The method accordingto claim 33, wherein step d) comprises: a) detecting a first signal fromthe hybridization of the detection probe with the first capture probeand a second signal from the hybridization of the detection probe withthe second capture probe; and b) calculating a ratio between the firstsignal and the second signal, wherein a ratio of at least 2 indicatesthat the biological sample contains DNA that is homozygous for thenormal connexin 26 gene, a ratio of no more than 0.5 indicates that thebiological sample contains DNA that is homozygous for the E47X mutation,and a ratio of greater than 0.5 and less than 2 indicates that thebiological sample contains DNA that is heterozygous for the E47Xmutation.
 40. The method of claim 32, wherein before step a), the DNAcontained in the biological sample is amplified using a pair of primers.